We are studying monocular and polymorphonuclear phagocytes in infection and inflammation. The studies proposed focus on functions of plasma membrane transporters, and of receptors for nucleotide and extracellular matrix proteins on these cells. The project has three aims: #1. To identify by expression cloning in Xenopus oocytes the plasma membrane receptors on mononuclear phagocytes for UTP and for ATP. Mononuclear phagocytes express two distinct types of surface receptors for nucleotide. 1. AP2u-type receptor that signals a rise in cytosolic calcium ([Ca+]i}, when it binds UTP. While UTP dose not signal any known effector function, it potentiates these cells effector system (e.g., H2O2 secretion). 2. A P3Z type receptor that responds to ATP-by opening a pore in the cells' plasma membrane. This pore is permeable to molecules of up to 900 daltons. It has been suggested that the pore is formed by the insertion of a gap junction protein, into the macrophage surface. We have expressed in Xenopus oocytes the P2u and P2Z-like activities of J774 macrophage receptors and are cloning the cDNAs encoding these activities. #2. To determine whether inhibitors of macrophage organic anion transport increase the efficacy of antibiotics against Mycobacterium, Avium complex, Legionella pneumophila, and Listeria monocytogenes, in human monocytes and macrophage to identify the transporter molecule by expression cloning, and to determine whether leukotriene C4 is an endogenous substrate for this transporter. We have discovered a pathway by which mouse macrophage secrete beta-lactam and quinoline antibiotics from their cytoplasm into the extracellular medium. (We have evidence that a similar pathway exists in human macrophage.) By this means, macrophage decrease the efficacy of these drugs against Listeria growing within their cytoplasm. We have discovered the gemfibrozil (GFZ) blocks this secretory pathway and increases the efficacy of quinoline antibiotics against intracellular listeria in Vitro. We will determine whether GFZ enhances the efficacy of quinoline antibiotics against Mycobacterium, Avium complex, Legionella pneumophila, and Listeria monocytogenes in human monocytes, and macrophage. We will identify the transporter molecule by expression cloning and determine whether it mediates the secretion of exogenous and endogenous substances from macrophage. #3 To characterize the mechanisms by which plasma membrane receptors of phagocytic phagocytes to crawl under some circumstances, and to become sessile under others. Whether they crawl or adhere appears to be a function of both the identify of the chemokine/chemoattractant and the substrate on, or in the which the cells are located. By blocking specific adhesion promoting receptors on phagocytes we have changed their response to one chemokine (tumor necrosis factor) from adhesion to migration through fibrin gel. We are exploring the mechanisms that control cell migration, and adhesion; and the effects of different matrix proteins on these processes.
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